Quote of the Week: Comparing Screen and Array Datasets

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays [in yeast] across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses.

Yeger-Lotem et al. (2009) Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nature Genetics. 41(3):316-323. PubMed ID: 19234470.

A Look at the DRSC: Main Lab

Breaking Paper--Control of Cell Size

On my desk today? Sims et al. (2009) PDGF/VEGF signalling controls cell size in Drosophila. Genome Biology. PubMed ID 19216764.  As of this posting, abstract viewable on-line and free full text downloadable as provisional PDF.

The paper focuses on cell size-related results from a genome-wide screen performed at the DRSC (see also Boutros et al. 2004 Science 303:832-835). 

Protocol Notes:  Screen done with S2R+ cells. Image data were put through an automated image analysis to determine average cell size (total monolayer cell area divided by the number of DAPI-positive nuclei counts). With by-eye follow up to check for phenotypes like cell adhesion effects that were not of interest for this study but might also score as positives. Network analysis using FLIGHT. Hit validation using two independent amplicons and a related but different image-based analysis.

Quote of the Week--Act III, Scene 2

Poor harmless fly,

That with his pretty buzzing melody

Came here to make us merry!

William Shakespeare, Titus Andronicus

Web Wednesday--50th ADRC Abstracts On-Line

Today's web feature is the Abstract Planner, now on-line in advance of the upcoming 50th Annual Drosophila Research Conference in Chicago, IL, USA.  Search away!  And remember, in an effort to go green, this year's conference booklets will not have full abstracts.

Breaking Report--Ecdysone & Death

On my desktop today?  

A report from Chittaranjan et al. (2009) Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi. Free online access from PLoS Genetics. (Figure show is from this report.)

Like the report from Rogers et al. that I blogged about in a previous post (Little Screens, Big Results), these authors focused their screen on a set of candidate genes. In this case, a set of 460 candidates. To do this, the authors generated dsRNAs for each candidate and followed up with a second dsRNA for screen hits.

Chitteranjan et al. performed what is essentially two screens. One focused on identification of pro-death and pro-surivival factors. And the other on "death related effects" dependent upon or independent of ecdysone treatment.

Protocol notes:  Their screen was done on ecdysone-treated l(2)mbn cells. Death and survival monitored with what I think of as an old-school (but reliable) approach, a trypan blue exclusion assay. And they took a closer look at cell death with DAPI staining and TUNEL analysis.

Some thoughts:  Reading another report like this focused on a defined set of candidate genes has me excited for something the DRSC has just begun to offer:  synthesis of custom sets of dsRNAs in 96-well format (based on existing amplicons).  

This made sense as (1) we're expert at doing IVTs at high-throughput, including quality analysis and troubleshooting, and have ready access to our collections of PCR amplicons used as templates for the dsRNA. And (2) the increasing interest in the community to screen a custom-curated sets of genes.  

We just successfully completed a pilot project and given the right lead-time, can now accommodate more requests. 

Please contact me if you're interested to learn more about the service. 

Quote of the Week--Looking Forward

Today's quote comes from a recent News & Views on two papers that describe multiple, simultaneous disruption of gene functions. We here at the DRSC are seeing an increase in the number of studies that combine multiple high-throughput approaches and/or disruption under multiple contexts, working towards understanding networks at a systems level.

The authors conclude in their review of the work that:

The analysis of multiple mutants and multiply perturbed cells provides crucial information for reconstructing the ‘wiring diagram’ of the cell.

Quote from:  Giaever & Nislow (2009) Knocking sense into regulatory pathways. Nature Biotechnology 27(2):149-150.

Papers discussed in this News & Views:

Bakal, C. et al.  (2008) Phosphorylation networks regulating JNK activity in diverse genetic backgrounds. Science 322, 453–456.  Featuring work performed at the DRSC.

Capaldi, A.P. et al. (2008) Structure and function of a transcriptional network activated by the MAPK Hog1. Nat. Genet. 40, 1300–1306.

Poison Control

On my desktop today is a paper from Muniz Ortiz et al. (2009) Investigating Arsenic Susceptibility from a Genetic Perspective in Drosophila Reveals a Key Role for Glutathione Synthetase. Toxilogical Sciences 107(2), 416–426. PubMed ID 18779381.

Among other things, the authors present evidence that RNAi-based knockdown of glutathione synthetase (GS) in S2 cells enhances cell sensitivity to arsenite. Raising the question in my mind, Could other factors in the pathway be revealed in a genome-scale RNAi screen?  

But why do we care? The authors point out that arsenic is a contaminant of drinking water. And different human populations (and the authors show, different Drosophila populations) respond differently to various levels of contamination, suggesting a genetic link to sensitivity. 

Thus, gaining a better understanding of genetic links to arsenite sensitivity may have important implications for water policy decisions and human health.

Quote of the Week: Cell Division

From a discussion of the 1838 cell theory proposed by Shcleiden & Schwann that appears in The Cell Cycle: An Introduction (1993) Murry & Hunt (W.H. Feeman & Co., New York):

Cell division is the only path to immortality. Nondividing cells can live for as long as a hundred years, but they always eventually die.

Upcoming Meeting

3rd Annual Arthropod Genomics Symposium, Frontiers in Arthropod Genomics. June 11 – 14, 2009, in Kansas City, USA. Keynote Speakers: F.C. Kafotis & W.M. Gelbart.

Little Screens, Big Results

What am I reading today?  Rogers et al. (2009) The SCF[Slimb] ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication. JCB 184(2):225-239. PubMed ID 19171756.

Why? This paper reports the results of a small-scale Drosophila RNAi screen the authors based on a set of candidate genes; namely, SCF-class ubiquitin ligases. The authors designed and generated dsRNAs for RNAi experiments targeting 58 genes in that family, and conducted their screen on those 58 targets.

By focusing in on a small set of candidates, the researchers were able to go into enviable depth in at least these two ways: first, they did not one but two sequential primary assays; and second, their further analysis of hits included to assess knockdown at the protein level for those genes for which antibodies were available.

Technical notes:  the authors used EGFP and mCherry markers, live cell imaging, and induction of cell cycle arrest (S phase, G2 phase or mitosis) with drug treatments. Imaging with S2 cells plated on Con-A coated glass-bottom 6-well dishes. Analyses included FACS, fluorescence microscopy and EM.

Web Wednesday--Distractions

Looking to be entertained? Tired of explaining why genetics is relevant to your relatives? Today's Web Wednesday focus is the Routes site out of the UK. It's genetics updated, with reality-style drama videos and a game that lets you create a creature, send it out into the ether and find out how well your genes fare in the population.

dsRNA Transport & Systemic RNAi

What am I reading today? Some reports on SID-1, an RNA transporter from C. elegans.

Feinberg & Hunter (2003) Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301: 1545-1547. PubMed ID 12970568.

Shih et al. (2009) The SID-1 double-stranded RNA transporter is not selective for dsRNA length. RNA. DOI 10.1261/rna.1286409. PubMed ID 19155320.

Jose et al. (2009) Export of RNAi silencing for C. elegans does not require the RNA channel SID-1. PNAS.  PubMed ID 19168628.

Why am I reading these? (1) Introduction of SID-1 into Drosophila cells can sensitize them to dsRNA treatment, presumably by facilitating uptake. (2) In C. elegans, RNAi effects spread systemically, a phenomenon not observed in Drosophila. But it looks like SID-1 is not the whole answer when it comes to systemic spread.

Related of interest:

Hunter et al. (2006) Systemic RNAi in Caenorhabditis elegans. CSH Symp Quant Biol. 71:95-100. PubMed ID 17381285.

Tomoyasu et al. (2008) Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium. Genome Biol 9(1):R10. PubMed ID 18201385. Free full text at Genome Biology.